Within a biological cell, levels of expression and activity of proteins are regulated by a subset of typically about 10% of the proteins, the subset that is dedicated to cell communications and control. This subset is referred to herein as “cell signaling proteins”. One of the largest classes of such cell signaling proteins is a class of enzymes called protein kinases. Protein kinases control other proteins by catalyzing their phosphorylation, a process that is reversible by protein phosphatases. Virtually all cell signaling proteins are either protein kinases or regulators of protein kinases or their substrates. Protein kinases often operate within signaling pathways that are further integrated into signaling networks.
Generally, such signaling pathways and networks govern and coordinate all cellular functions, including cell structure, metabolism, reproduction, adaptation, differentiation and death, for example.
Moreover, protein kinases appear to be disproportionately linked to cell diseases. For example, approximately half of the hundred or so identified cancer-inducing genes, or “oncogenes”, encode protein kinases, and the remaining half appear to encode proteins that either activate kinases or are phosphorylated by kinases. In addition, over 400 human diseases, such as cardiovascular disease, diabetes, arthritis and other immune disorders, and Alzheimer's disease and other neurological disorders, for example, have been linked to defective signaling through protein kinases.
If protein kinases and their respective signaling pathways and networks can be identified and understood, then monitoring of the kinases could feasibly be used to obtain a molecular diagnosis of a disease condition. In addition, if a particular problematic protein kinase in a diseased cell can be identified, it may be possible to inhibit either the problematic kinase or its downstream effectors, to effectively block the improper proliferative signaling, or even to initiate apototic processes leading to programmed death of the diseased cells such as tumor cells for example.
Therefore, it is particularly important to identify not only the protein kinases and other cell signaling proteins themselves, but more importantly, the signaling pathways and networks in which they operate.
Identification of the kinases themselves is presently being achieved through various genome sequencing projects, and virtually all of the human kinases are expected to be identified within the next year.
However, existing techniques are not suitable for identifying signaling pathways and networks of cell signaling proteins. Most protein kinases appear to be activated as a consequence of either their own phosphorylation by upstream kinases, or by autophosphorylation (i.e. self-phosphorylation), however, nucleic acid-based techniques such as those used for detection of gene expression cannot be used to monitor post-translational events such as phosphorylation.
Conversely, conventional measurement techniques that are capable of differentiating between phosphorylated and dephosphorylated states of proteins, such as the standard two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) technique of Dr. Patrick O'Farrell for example, have experienced great difficulty in detecting protein kinases, which are typically present at very minute levels in a cell compared to other proteins. For example, public databases exist containing identifications of over a thousand different proteins on two-dimensional gel maps, but containing identifications of scarcely more than a dozen of the estimated two thousand or so protein kinases that are thought to be encoded by the human genome.
Recent modifications of the two dimensional SDS-PAGE technique involving the Western blotting procedure followed by detection using a single monoclonal anti-kinase antibody per blot, for example, have been attempted. However, the recovery of most protein kinases from the first dimension gel is typically less than 10%, with the result that 90% or more of protein kinases do not enter the second dimension gel and are therefore unresolved. Accordingly, these recent modified techniques are typically able to detect only four or five of the several hundred protein kinases that are expected to be present in a given cell.
Due to the above difficulties in obtaining measurements of levels and phosphorylation states of cell signaling proteins such as kinases, few, if any, analytical techniques exist for the analysis of kinase measurements to yield information about the signaling pathways and networks in which the kinases operate.
However, the inventor of the invention disclosed and claimed herein has recently invented a new and useful method for detection of multiple cell signaling proteins, such as multiple kinases or multiple kinase substrates (i.e. proteins that are phosphorylated by kinases), whereby the presence and phosphorylation states of a large number of kinases and/or kinase substrates may be measured in a single sample.
Thus, with the advent of this new experimental measurement technique, there is a need for new analytical techniques for analyzing cell signaling protein measurements, to identify particular cell signaling proteins that are associated with one another in common signaling pathways.